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Central catecholamine receptor blocking actions of BE-2254 (‘HEAT’): Comparison with chlorpromazine and haloperidol
European Journal of Pharmacology, 32 (1975) 279--286 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
C E N T R A L C A T E C H O L A M I N E R E C E P T O R BLOCKING ACTIONS OF BE-2254 ('HEAT'): COMPARISON WITH CHLORPROMAZINE AND HALOPERIDOL BRADLEY V. CLINESCHMIDT, A. BARBARA PFLUEGER, PATRICIA R. BUNTING, JODIE C. McGUFFIN and RUBY J. BALLENTINE Merck Institute for Therapeutic Research, West Point, Pennsylvania 19486, U.S.A. Received 27 January 1975, revised MS received 14 March 1975, accepted 21 March 1975
B.V. CLINESCHMIDT, A.B. PFLUEGER, P.R. BUNTING, J.C. McGUFFIN and R.J. BALLENTINE, Central catecholamine receptor blocking actions of BE-2254 ('HEAT'): comparison with chlorpromazine and haloperidol, European J. Pharmacol. 32 (1975) 279--286. BE-2254, 2-[/3-(4-hydroxyphenyl)-ethylaminomethyl]-tetralone, (EDs0 -- 3.4 mg/kg i.p.) was about equal to chlorpromazine (EDs0 = 4.4) as an antagonist of central noradrenergic receptor stimulation produced by clonidine (enhancement of the flexor reflex in spinalized rats). Haloperidol and phentolamine had essentially no effect at 9 mg/kg i.p. At higher doses, BE-2254 also blocked central receptors for dopamine. BE-2254 (EDs 0 = 55.0 mg/kg i.p.) was considerably less active than haloperidol (EDs 0 = 0.11 ) or chlorpromazine (ED s 0 = 1.42) as an antagonist of apomorphine-induced gnawing in rats. However, BE-2254 (EDs 0 = 16.4 mg/kg i.p.) more closely approximated haloperidol (EDs 0 = 0.54) and chlorpromazine (EDs 0 = 3.1) in eliciting rotational movements in mice with a unilateral striatal lesion. BE-2254 (EDs0 = 1.6 mg/kg i.p.) antagonized amphetamine-stimulated locomotor activity in mice, providing additional evidence that the compound can significantly inhibit central catecholamine receptor-mediated responses. It is concluded that BE-2254 blocks receptors for both norepinephrine and dopamine in the CNS -- the former at lower doses than the latter. Although additional studies are required to more clearly define the selectivity of BE-2254, it appears that this agent is useful for producing blockade of central receptors for norepinephrine. Norepinephrine receptor antagonist Dopamine receptor antagonist
BE-2254 ('HEAT') Chlorpromazine
1. I n t r o d u c t i o n Despite the r e a s o n a b l y p o t e n t b l o c k i n g action of BE-2254 ('HEAT'; 2-[3-(4-hydroxyp h e n y l ) - e t h y l a m i n o m e t h y l ] - t e t r a l o n e ) o n peripheral a-adrenergic r e c e p t o r s ( B e n t h e et al., 1 9 7 2 ) , the c o m p o u n d has n o t been e x a m i n e d to a n y e x t e n t for possible central catechola m i n e r e c e p t o r b l o c k i n g activity. In fact, t h e o n l y i n f o r m a t i o n available is o f a r a t h e r incid e n t a l nature, c o m i n g f r o m a r e c e n t s t u d y b y Y e h u d a and W u r t m a n (1974). We have, a c c o r d i n g l y , c o m p a r e d B E - 2 2 5 4 with c h l o r p r o m a z i n e and h a l o p e r i d o l in some p h a r m a c o l o g i c a l test p r o c e d u r e s useful f o r asc e r t a i n i n g b l o c k a d e o f central r e c e p t o r s f o r
Haloperidol
Amphetamine
n o r e p i n e p h r i n e and d o p a m i n e . I n h i b i t i o n of the e n h a n c e m e n t o f the flexor reflex prod u c e d b y c l o n i d i n e in the spinal rat ( A n d ~ n et al., 1 9 7 0 ) was e m p l o y e d as a measure o f central noradrenergic receptor antagonism. B l o c k a d e o f central r e c e p t o r s f o r d o p a m i n e was assessed, using elicitation of a s y m m e t r i c m o v e m e n t s in mice with a unilateral striatal lesion (Lotti, 1 9 7 1 ) and a n t a g o n i s m o f apom o r p h i n e - i n d u c e d gnawing in rats (Ernst, 1 9 6 7 ) . B E - 2 2 5 4 was a d d i t i o n a l l y evaluated for its c a p a c i t y to impair central catechola m i n e - m e d i a t e d transmission b y d e t e r m i n i n g its ability to a n t a g o n i z e a m p h e t a m i n e - s t i m u lated l o c o m o t o r activity in mice.
280
B.V. CLINESCHMIDTET AL.
2. Materials and methods
of 10 rats/treatment was used in making the above calculation.
2.1. Flexor reflex 2.3. Lesioned mouse Female CFE Carworth rats weighing 1 6 0 200 g were anesthetized with ether and the spinal cord transected in the mid-thoracic region. Ether was discontinued, and the animals were allowed 4--5 hr to recover prior to administering any drug treatments. Pretreatment of the spinalized rats was performed 30 min prior to treatment with clonidine, 1.2 mg/kg i.p. The flexor reflex was evoked by pinching the hind feet with forceps 30, 60, 120 and 180 min after administration of clonidine. The strength of the reflex was scored from 0 (absent) to +3 (vigorous). Rats not treated with clonidine were invariably scored as 0, thus obviating the n e e d for including this group in calculation of % inhibition. Percent inhibition was, therefore, calculated as follows: (vehicle + clonidine) -- (drug + clonidine)
100 ×
(vehicle + clonidine)
The sum of the scores obtained with groups of 16 rats/treatment was used in making the calculation given above. At each pretreatment dose level (1, 3 or 9 mg/kg), BE-2254, chlorpromazine and haloperidol were tested concurrently under random and blind conditions. For additional details see fig. 1, see Results.
2.2. A p o m o r p h i n e antagonism 1 hr after pretreatment, the rats (female CFE Carworth; 160--200 g) were treated with apomorphine (3 mg/kg s.c.) and placed in individual cages. I hr later the animals were scored under blind and randomized conditions from 0 (absent) to +3 (pronounced) for stereotyped gnawing. Percent inhibition of the apomorphine-induced response was calculated as follows (v = vehicle): [(v + apomor.)-100 ×
(v + v)] -- [(drug + apomor.)
-- (v + v)]
(v + a p o m o r . ) -- (v + v)
The sum of the scores obtained with groups
Carworth CFI female mice weighing 18-22 g were prepared with a unilateral suction lesion of the striatum as described by Lotti (1971). The animals were allowed at least 14 days for recovery prior to initiating drug testing. The number of mice exhibiting postural asymmetries (deviations or circling) contralateral to the lesioned side was ascertained, using 10 mice per dose level of each compound. The mice were observed 1, 2 and 3 hr after injection. These studies were also performed using r a n d o m and blind conditions.
2.4. Mouse locomotor activity L o c o m o t o r activity was assessed with 'donut'-shaped photocell chambers, consisting of a circular runway with a central light source and 6 15hotocells equally spaced around the outer wall of the runway. Each unit was 6-3/4 inches in overall diameter with the runway being 2-1/2 inches wide. Counts resulting from interruption of the light beams were recorded with Sodeco counters (25 impulses/sec). Background noise was supplied with a Grason Stadler Noise Generator. Experiments were conducted with one mouse (CFI female) per activity chamber. Mice with different treatments were assigned to a particular chamber on a totally randomized basis, and pretreated and control animals were tested concurrently. Activity counts were recorded for 1 hr, beginning 15 min after the final treatment.
2. 5. Data analysis EDs 0 values and 95% confidence intervals were estimated via regression analysis. To test for differences among treatment groups, Student's t-test was employed with a p value of <0.05 (2-tailed) considered as significant. Data from the locomotor activity studies were
BE-2254 AND CATECHOLAMINE RECEPTOR BLOCKADE
transformed into the square roots of the counts prior to applying the t-test. This was necessary, since, b o t h in control mice and those treated with amphetamine, the frequency distribution of the motor activity counts was found to be skewed in the positive direction, i.e. higher counts being farther away from the mean than lower counts.
281
,oo
L
I e, o ~.
5
, • BE-2254 o CPZ h Holoperidol
L~ 5O
2. 6. Drugs The doses of all c o m p o u n d s are in terms of the base with the exception of amphetamine. All c o m p o u n d s were administered using 1% methylcellulose as vehicle except for amphetamine which was dissolved in distilled water. Haloperidol, chlorpromazine hydrochloride, BE-2254 and phentolamine hydrochloride were, respectively, generous gifts from McNeil Laboratories (Fort Washington, Pa.), Smith Kline Laboratories (Philadelphia, Pa.), Beiersdorf, A.G. (Hamburg, G e r m a n y ) a n d Ciba-Geigy (Summit, New Jersey). D-Amphetamine sulfate and apomorphine hydrochloride were obtained from the Medicinal Chemistry Section of Merck and Co. (West Point, Pa.).
3. Results
3.1. Antagonism of clonidine-induced enhancement o f the flexor reflex in the spinalized rat At the time of the first test, i.e. 30 min after treatment with clonidine, BE-2254 produced its greatest inhibitory effect (EDs 0 = 3.4 mg/kg i.p.) (fig. 1). The inhibitory action of BE-2254 subsided only gradually over time with the EDs 0 values at 60, 120 and 180 min being respectively, 3.6, 3.7 and 3.8 mg/kg. In contrast, the antagonistic action of chlorpromazine developed slowly with the ED50 values 30, 60, 120 and 180 min after treatment with clonidine being, respectively, 7.5, 5.1, 4.4 and 4.4 mg/kg i.p. (fig. 1). Haloperidol was essentially inactive at all time inter° vals and at all doses examined.
8 ~5
0
A I
3
9
mg/kg i.p.
Fig. 1. Antagonism of clonidine-induced enhancement of the flexor reflex in the spinalized rat. Pretreatment with vehicle, BE-2254, chlorpromazine (CPZ) or haloperidol was performed 30 min prior to treatment with clonidine, 1.2 mg/kg i.p. At each of the 3 pretreatment dose levels, the compounds were tested concurrently under random and blind conditions. The strength of the flexor reflex was determined 30, 60, 120 and 180 rain after administering clonidine. Values plotted in the figure are at time of greatest antagonism of clonidine, as follows: BE-2254 -- 30 rain, c h i o r p r o m a z i n e - 120 min, and haloperidol -- 60 rain after injecting clonidine. EDs0 values estimated from these data were: BE-2254, 3.4 (95% confidence interval = 2.9--3.9), chlorpromazine = 4.4 (4.3--4.6) and haloperidol >9 mg/kg i.p.
Phentolamine was tested at one dose level, 9 mg/kg i.p. 30 min prior to treatment, for inhibition of clonidine-induced enhancement of the flexor reflex. 10 rats received phentolamine + clonidine and a second 10 got vehicle + clonidine. Total scores (maximum total possible = 3 × n = 30) for phentolamine + clonidine at 30, 60, 120 and 180 min after injecting clonidine were 25, 26, 24 and 22, while in the vehicle-pretreated animals the total scores equaled 25, 27, 26 and 25. Phentolamine was, obviously, ineffective.
282
B.V. CLINESCHMIDT ET AL
z
°iO0
50 c.) c~
_z
BE-2
HALOPERI D
8 o
I
I
I
I
I
I
0.05
0.I
0.5
I
5
I0
50
Ioo
mg/kg i.p.
Fig. 2. Inhibition of apomorphine-induced gnawing in rats. 1 hr following pretreatment, the rats received apomorphine (3 mg/kg s.c.). 1 hr later, the animals were observed for the presence and degree of stereotyped gnawing. Details are given under Materials and methods. EDs 0 values estimated from these data were: haloperidol = 0.11 (95% confidence interval 0.11--0.11), chlorpromazine (CPZ) = 1.42 (1.24--1.37) and BE-2254 = 55.0 (48.2--62.2). TABLE 1 Elicitation of posturai asymmetries in mice with a unilateral striatal lesion. Treatment (mg/kg i.p.)
Methocel BE-2254
Methocel Haioperidol
Methocel Chlorpromazine
No.* contralateral asymmetries/No, tested 1 hr
2 hr
3 hr
(8.9) (13.3) (20.0) (30.0)
1/10 2/10 3/10 6/10 9/10 E D s 0 : 1 6 . 4 (12.4--21.6)**
1/10 0/10 1/10 3/10 4/10 > 30
0/10 0/10 0/10 0/10 0/10 > 30
(0.29) (0.47) (0.75) (1.2)
0/10 1/10 0/10 5/10 5/10 EDs 0 : --
0/10 0/10 2/10 7/10 9/10 0.64
0/10 2/10 3/10 8/10 9/10 0.54 (0.33--0.88)**
(1.5) (2.25) (3.38) (5.06)
0/10 1/10 1/10 5/10 10/10 EDs0:3.4
0/10 0/10 1/10 5/10 10/10 3.1 (2.1--4.6)**
0110 0/10 0/10 5/10 10110 3.4
* Deviations or circling to side opposite the lesion. Observations for asymmetries were carried out under random and blind conditions at 1, 2 and 3 hr after treatment. ** 95% confidence interval, enclosed in parentheses, is given for time of maximum effect.
BE-2254 A N D C A T E C H O L A M I N E R E C E P T O R B L O C K A D E
3.2. Antagonism gnawing in rats
of
283
ble 1). The action of BE-2254 was rather transient, whereas that of haloperidol developed gradually with time. Chlorpromazine appeared to be a b o u t equally effective at 1, 2 or 3 hr post injection.
apomorphine-induced
BE-2254 was considerably less p o t e n t (EDs0 = 55.0 mg/kg i.p.; 95% confidence interval = 48.2--62.2) than either chlorpromazine (EDs 0 = 1.42; 1.24--1.37) or haloperidol (EDs 0 = 0.11; 0.11--0.11) as an antagonist of apomorphine (fig. 2).
3. 4. Inhibition o f amphetamine-stimulated locomotor activity Stimulation of l o c o m o t o r activity in mice by amphetamine was virtually completely blocked by 5 mg/kg of BE-2254 or 0.5 mg/kg of haloperidol (table 2). BE-2254 (EDs0 = 1.6 mg/kg) was about 1/10th as potent as haloperidol (EDs0 = 0.18 mg/kg) as an antagonist of amphetamine.
3.3. Elicitation o f postural asymmetries in lesioned mice All three c o m p o u n d s elicited deviations or circling contralateral to the lesioned side (taTABLE 2
A n t a g o n i s m o f a m p h e t a m i n e - s t i m u l a t e d l o c o m o t o r activity in mice.
Pretreatmentt (mg/kg i.p.)
Treatmenttt (mg/kg s.c.)
Vehicle Vehicle BE-2254
(5) (5) (2.5) (2.5) (1.25) (1.25) (0.625) (0.625)
Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine
(0.5) (0.5) (0.25) (0.25) (0.125) (0.125) (0.0625) (0.0625)
Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine
Vehicle Vehicle Haloperidol
Counts/1 hr (~-± S.D. of square roots)
(3) (3) (3) (3) (3)
(3) (3) (3) (3) (3)
42.2 84.9 *27.3 **29.5 *34.9 **45.7 38.0 **65.6 36.6 *'74.1
± ± ± ± ± ± ± ± ± ±
9.8 15.8 11.4 12.1 12.4 20.9 11.3 17.2 14.7 20.0
37.5 66.0 "16.0 *'21.0 "18.6 **29.8 *26.0 **43.2 *26.5 59.6
± ± ± ± ± ± ± ± ± ±
6.3 10.5 7.7 9.8 5.8 9.0 8.2 12.2 8.2 14.4
A m p h e t a m i n e ++ % Inhibition stimulated activity
42.7 2.2
94.6
10.8
74.6
27.6
35.3
37.5
12.2 EDs 0 = 1.61 (1.19--2.17) +
28.5 5.0
82.5
11.2
60.7
17.2
39.6
33.1 EDs 0 = 0.18 (0.16--0.19) +
t tt + ++ * **
n = 2 0 / g r o u p ; 30 (BE-2254) or 90 (Haloperidol) rain before t r e a t m e n t . 15 rain before i n t r o d u c t i o n into " d o n u t " - s h a p e d p h o t o c e l l units. 95% c o n f i d e n c e interval. ( P r e t r e a t m e n t + a m p h e t a m i n e ) -- ( p r e t r e a t m e n t + vehicle). C o m p a r e d with vehicle + vehicle C o m p a r e d w i t h vehicle + a m p h e t a m i n e ~ P < 0.05 (2-tailed t-test).
284
4. Discussion
In addition to stimulating s-receptors for norepinephrine in peripheral tissues (Hoefke and Kobinger, 1966), clonidine also stimulates receptors in the CNS having characteristics similar to the peripheral s-receptor (And~n et al., 1970; Kobinger and Walland, 1971; Schmitt et al., 1971). And~n (1970) has summarized the rather extensive evidence indicating that activation of noradrenergic receptors in the spinal cord leads to an enhancement of the flexor reflex. Antagonism of the increase in the flexor reflex elicited by clonidine thus provides a means for identifying centrally acting antagonists of receptors for norepinephrine. BE-2254 and chlorpromazine were effective in this regard, whereas phentolamine and haloperidol were not. Clonidine's action on the flexor reflex can also be inhibited somewhat with the s-blockers phenoxybenzamine and yohimbine, b u t high (~>10 mg/kg) doses are required (unpublished observations). Considering the differences between the experimental procedures, it seems rather interesting that our findings agree closely with results obtained from microiontophoretic studies on neurons in the brain stem of the cat. Chlorpromazine (Bradley et al., 1966) specifically antagonizes the excitatory action of norepinephrine on brain stem neurons, while phentolamine and phenoxybenzamine are essentially ineffective (Boakes et al., 1971). Apomorphine elicits stereotyped behavior, including compulsive gnawing, by a direct agonistic effect on central receptors for dopamine (Ernst, 1967; And~n et al., 1967). Inhibition of apomorphine-induced gnawing by BE-2254 suggested, therefore, an ability to act as a central dopamine receptor antagonist. Like the widely accepted antagonists of dopamine, chlorpromazine and haloperidol, BE2254 also produced postural asymmetries in mice with a unilateral lesion in the striatum. These findings were somewhat unexpected, since such an action of BE-2254 could hardly
B.V. C L I N E S C H M I D T ET AL.
have been predicted from the compound's chemical structure. An interesting difference was noted in the relative potency of BE-2254 in the two tests for dopamine receptor antagonism. BE-2254 was about 500 times less potent than haloperidol in the apomorphine antagonism test, but only approximately 30 times less active in the postural asymmetry test. Several explanations could be advanced, although two seem most likely. First of all, it could simply reflect a species difference, since the rat was used in one procedure and the mouse in the other. The time course of the blocking effect of BE-2254 on receptors for dopamine might also provide at least a partial explanation. As shown in the lesioned mouse, BE-2254's action was transient, falling off greatly between 1 and 2 hr post injection. Antagonism of apomorphine-evoked gnawing was determined only at one time, 2 hr after pretreatment with BE-2254 or one of the other compounds. Therefore, assuming transient dopamine receptor blockade in the rat as well, the effect of BE-2254 may have greatly subsided by 2 hr following administration, while the longer acting haloperidol was still quite effective. In any event, our observations clearly demonstrate the central dopamine receptor blocking action of BE-2254, and it is not clear why Yehuda and Wurtman (1974) did not detect this effect of BE-2254 in the rat, using either the apomorphine stereotypy test or the rotational behavior model. Although BE-2254 resembles chlorpromazine, inasmuch as it appears to block central receptors for either norepinephrine or dopamine, BE-2254 also differs importantly from chlorpromazine. BE-2254 has little, if any, peripheral (and presumably central) antiserotonin activity (Gothert et al., 1973; Baumgarten et al., 1972}. Moreover, unlike chlorpromazine, BE-2254 does not cause any significant dilation of the mouse's pupil in doses up to 30 mg/kg i.p. (unpublished observations), suggesting a lack of any substantial anticholinergic activity. As the data reported herein would indicate, BE-2254 is a b o u t comparable
BE-2254 AND CATECHOLAMINE RECEPTOR BLOCKADE
to chlorpromazine as an antagonist of central receptors for norepinephrine, but it is a weaker agent with respect to receptors for dopamine. Considering the above, BE-2254 may prove to be a useful pharmacological agent for producing blockade of central receptors for norepinephrine. It is generally agreed that amphetamine stimulates locomotor activity in mice and rats by releasing catecholamines from monoaminecontaining neurons in the CNS. However, there is no agreement concerning the relative importance of dopamine and norepinephrine, and findings reported by several authors suggest that both amines are involved (Corrodi et al., 1970; Svensson, 1970; Rolinski and ScheelKriiger, 1973). Inhibition by BE-2254 of amphetamine-stimulated locomotor activity in the mouse provides additional indirect evidence that this compound antagonizes central catecholamine receptor-dependent responses. Based on the evidence provided in this study, it seems likely that BE-2254 antagonizes amphetamine by virtue of its receptor blocking action, although there is no evidence for excluding other possible mechanisms, e.g. inhibition of catecholamine release. To summarize, indirect evidence has been obtained indicating that BE-2254 administered systemically blocks central receptor sites for catecholamines and impairs catecholamine receptor-mediated responses. The norepinephrine receptor blocking action of BE2254 is roughly comparable to that of chlorpromazine, whereas BE-2254 is considerably less active than chlorpromazine or haloperidol as an antagonist of receptors for dopamine. Although additional evidence is admittedly needed in order to establish the selectivity of BE-2254, it appears that this agent is useful for producing blockade of central receptors for norepinephrine.
References And~n, N.-E., 1970, Effects of amphetamine and some other drugs on central catecholamine mecha-
285
nisms, in: International Symposium on Amphetamines and Related Compounds, eds. E. Costa and S. Garattini (Raven Press, New York) p. 447. And~n, N.-E., H. Corrodi, K. Fuxe, B. HSkfelt, T. H6kfelt, C. Rydin and T. Svensson, 1970, Evidence for a central noradrenaline receptor stimulation by clonidine, Life Sci. 9, Part I, 513. And~n, N.-E., A. Rubenson, K. Fuxe and T. HSkfelt, 1967, Evidence for dopamine receptor stimulation by apomorphine, J. Pharm. Pharmacol. 19, 627. Baumgarten, H.G., M. Gothert, H.G. Schlossberger and P. Tuchinda, 1972, Mechanism of pressor effect of 5,6-dihydroxytryptamine in pithed rats, Naunyn-Schmiedeb. Arch. Pharmakol. 274, 375. Benthe, H.F., M. Gothert and P. Tuchinda, 1972, Noradrenalin-antagonistische Wirkung verschiedener Phenylaethylamin- und PhenoxyaethylaminDerivate, Arzneim. Forsch. 22, 1468. Boakes, R.J., P.B. Bradley, N. Brookes, J.M. Candy and J.H. Wolstencroft, 1971, Actions of noradrenaline, other sympathomimetic amines and antagonists on neurones in the brain stem of the cat, Brit. J. Pharmacol. 41,462. Bradley, P.B., J.H. Wolstencroft, L. Hosli and G.L. Avanzino, 1966, Neuronal basis for the central action of chlorpromazine, Nature, London 212, 1425. Corrodi, H., K. Fuxe, A. Ljungdahl and S.-O. Ogren, 1970, Studies on the action of some psychoactive drugs on central noradrenaline neurones after inhibition of dopamine-~-hydroxylase, Brain Res. 24, 451. Ernst, A.M., 1967, Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats, Psychopharmacol. 10, 316. Gothert, M., P. Tuchinda and H.G. Baumgarten, 1973, Pressor effect of indolethylamines, European J. Pharmacol. 21,242. Hoefke, W. and W. Kobinger, 1966, Pharmakologische Wirkung des 2--(2,6--Dichlorophenylamino)2-Imidazolin-hydrochjorids, einer neuen, antihypertensiven Substanz, Arzneim. Forsch. 16, 1038. Kobinger, W. and A. Walland, 1971, Involvement of adrenergic receptors in central vagus activity, European J. Pharmacol. 16, 120. Lotti, V.J., 1971, Action of various centrally acting agents in mice with unilateral caudate brain lesions, Life Sci. 10, 781. Rolinski, Z. and J. Scheel-Kriiger, 1973, The effect of dopamine and noradrenaline antagonists on amphetamine-induced locomotor activity in mice and rats, Acta Pharmacol. Toxicol. 33, 385. Schmitt, H., H. Schmitt and S. F4nard, 1971, Evidence for an a-sympathometic component in the e f f e c t s of catapressan on vasomotor centres: antagonism by piperoxane, European J. Pharmacol. 14, 98.
286 Svensson, T.H., 1970, The effect of inhibition of catecholamine synthesis on dexamphetamine-induced central stimulation, European J. Pharmacol. 12, 161. Yehuda, S. and R.J. Wurtman, 1974, Paradoxical
B.V. CLINESCHMIDT ET AL thermoregulatory behavior in rats induced by (+) amphetamine: blockade by ~-adrenoceptor or dop amine receptor blocking agents, J. Pharm. Pharma col. 26, 210.
C E N T R A L C A T E C H O L A M I N E R E C E P T O R BLOCKING ACTIONS OF BE-2254 ('HEAT'): COMPARISON WITH CHLORPROMAZINE AND HALOPERIDOL BRADLEY V. CLINESCHMIDT, A. BARBARA PFLUEGER, PATRICIA R. BUNTING, JODIE C. McGUFFIN and RUBY J. BALLENTINE Merck Institute for Therapeutic Research, West Point, Pennsylvania 19486, U.S.A. Received 27 January 1975, revised MS received 14 March 1975, accepted 21 March 1975
B.V. CLINESCHMIDT, A.B. PFLUEGER, P.R. BUNTING, J.C. McGUFFIN and R.J. BALLENTINE, Central catecholamine receptor blocking actions of BE-2254 ('HEAT'): comparison with chlorpromazine and haloperidol, European J. Pharmacol. 32 (1975) 279--286. BE-2254, 2-[/3-(4-hydroxyphenyl)-ethylaminomethyl]-tetralone, (EDs0 -- 3.4 mg/kg i.p.) was about equal to chlorpromazine (EDs0 = 4.4) as an antagonist of central noradrenergic receptor stimulation produced by clonidine (enhancement of the flexor reflex in spinalized rats). Haloperidol and phentolamine had essentially no effect at 9 mg/kg i.p. At higher doses, BE-2254 also blocked central receptors for dopamine. BE-2254 (EDs 0 = 55.0 mg/kg i.p.) was considerably less active than haloperidol (EDs 0 = 0.11 ) or chlorpromazine (ED s 0 = 1.42) as an antagonist of apomorphine-induced gnawing in rats. However, BE-2254 (EDs 0 = 16.4 mg/kg i.p.) more closely approximated haloperidol (EDs 0 = 0.54) and chlorpromazine (EDs 0 = 3.1) in eliciting rotational movements in mice with a unilateral striatal lesion. BE-2254 (EDs0 = 1.6 mg/kg i.p.) antagonized amphetamine-stimulated locomotor activity in mice, providing additional evidence that the compound can significantly inhibit central catecholamine receptor-mediated responses. It is concluded that BE-2254 blocks receptors for both norepinephrine and dopamine in the CNS -- the former at lower doses than the latter. Although additional studies are required to more clearly define the selectivity of BE-2254, it appears that this agent is useful for producing blockade of central receptors for norepinephrine. Norepinephrine receptor antagonist Dopamine receptor antagonist
BE-2254 ('HEAT') Chlorpromazine
1. I n t r o d u c t i o n Despite the r e a s o n a b l y p o t e n t b l o c k i n g action of BE-2254 ('HEAT'; 2-[3-(4-hydroxyp h e n y l ) - e t h y l a m i n o m e t h y l ] - t e t r a l o n e ) o n peripheral a-adrenergic r e c e p t o r s ( B e n t h e et al., 1 9 7 2 ) , the c o m p o u n d has n o t been e x a m i n e d to a n y e x t e n t for possible central catechola m i n e r e c e p t o r b l o c k i n g activity. In fact, t h e o n l y i n f o r m a t i o n available is o f a r a t h e r incid e n t a l nature, c o m i n g f r o m a r e c e n t s t u d y b y Y e h u d a and W u r t m a n (1974). We have, a c c o r d i n g l y , c o m p a r e d B E - 2 2 5 4 with c h l o r p r o m a z i n e and h a l o p e r i d o l in some p h a r m a c o l o g i c a l test p r o c e d u r e s useful f o r asc e r t a i n i n g b l o c k a d e o f central r e c e p t o r s f o r
Haloperidol
Amphetamine
n o r e p i n e p h r i n e and d o p a m i n e . I n h i b i t i o n of the e n h a n c e m e n t o f the flexor reflex prod u c e d b y c l o n i d i n e in the spinal rat ( A n d ~ n et al., 1 9 7 0 ) was e m p l o y e d as a measure o f central noradrenergic receptor antagonism. B l o c k a d e o f central r e c e p t o r s f o r d o p a m i n e was assessed, using elicitation of a s y m m e t r i c m o v e m e n t s in mice with a unilateral striatal lesion (Lotti, 1 9 7 1 ) and a n t a g o n i s m o f apom o r p h i n e - i n d u c e d gnawing in rats (Ernst, 1 9 6 7 ) . B E - 2 2 5 4 was a d d i t i o n a l l y evaluated for its c a p a c i t y to impair central catechola m i n e - m e d i a t e d transmission b y d e t e r m i n i n g its ability to a n t a g o n i z e a m p h e t a m i n e - s t i m u lated l o c o m o t o r activity in mice.
280
B.V. CLINESCHMIDTET AL.
2. Materials and methods
of 10 rats/treatment was used in making the above calculation.
2.1. Flexor reflex 2.3. Lesioned mouse Female CFE Carworth rats weighing 1 6 0 200 g were anesthetized with ether and the spinal cord transected in the mid-thoracic region. Ether was discontinued, and the animals were allowed 4--5 hr to recover prior to administering any drug treatments. Pretreatment of the spinalized rats was performed 30 min prior to treatment with clonidine, 1.2 mg/kg i.p. The flexor reflex was evoked by pinching the hind feet with forceps 30, 60, 120 and 180 min after administration of clonidine. The strength of the reflex was scored from 0 (absent) to +3 (vigorous). Rats not treated with clonidine were invariably scored as 0, thus obviating the n e e d for including this group in calculation of % inhibition. Percent inhibition was, therefore, calculated as follows: (vehicle + clonidine) -- (drug + clonidine)
100 ×
(vehicle + clonidine)
The sum of the scores obtained with groups of 16 rats/treatment was used in making the calculation given above. At each pretreatment dose level (1, 3 or 9 mg/kg), BE-2254, chlorpromazine and haloperidol were tested concurrently under random and blind conditions. For additional details see fig. 1, see Results.
2.2. A p o m o r p h i n e antagonism 1 hr after pretreatment, the rats (female CFE Carworth; 160--200 g) were treated with apomorphine (3 mg/kg s.c.) and placed in individual cages. I hr later the animals were scored under blind and randomized conditions from 0 (absent) to +3 (pronounced) for stereotyped gnawing. Percent inhibition of the apomorphine-induced response was calculated as follows (v = vehicle): [(v + apomor.)-100 ×
(v + v)] -- [(drug + apomor.)
-- (v + v)]
(v + a p o m o r . ) -- (v + v)
The sum of the scores obtained with groups
Carworth CFI female mice weighing 18-22 g were prepared with a unilateral suction lesion of the striatum as described by Lotti (1971). The animals were allowed at least 14 days for recovery prior to initiating drug testing. The number of mice exhibiting postural asymmetries (deviations or circling) contralateral to the lesioned side was ascertained, using 10 mice per dose level of each compound. The mice were observed 1, 2 and 3 hr after injection. These studies were also performed using r a n d o m and blind conditions.
2.4. Mouse locomotor activity L o c o m o t o r activity was assessed with 'donut'-shaped photocell chambers, consisting of a circular runway with a central light source and 6 15hotocells equally spaced around the outer wall of the runway. Each unit was 6-3/4 inches in overall diameter with the runway being 2-1/2 inches wide. Counts resulting from interruption of the light beams were recorded with Sodeco counters (25 impulses/sec). Background noise was supplied with a Grason Stadler Noise Generator. Experiments were conducted with one mouse (CFI female) per activity chamber. Mice with different treatments were assigned to a particular chamber on a totally randomized basis, and pretreated and control animals were tested concurrently. Activity counts were recorded for 1 hr, beginning 15 min after the final treatment.
2. 5. Data analysis EDs 0 values and 95% confidence intervals were estimated via regression analysis. To test for differences among treatment groups, Student's t-test was employed with a p value of <0.05 (2-tailed) considered as significant. Data from the locomotor activity studies were
BE-2254 AND CATECHOLAMINE RECEPTOR BLOCKADE
transformed into the square roots of the counts prior to applying the t-test. This was necessary, since, b o t h in control mice and those treated with amphetamine, the frequency distribution of the motor activity counts was found to be skewed in the positive direction, i.e. higher counts being farther away from the mean than lower counts.
281
,oo
L
I e, o ~.
5
, • BE-2254 o CPZ h Holoperidol
L~ 5O
2. 6. Drugs The doses of all c o m p o u n d s are in terms of the base with the exception of amphetamine. All c o m p o u n d s were administered using 1% methylcellulose as vehicle except for amphetamine which was dissolved in distilled water. Haloperidol, chlorpromazine hydrochloride, BE-2254 and phentolamine hydrochloride were, respectively, generous gifts from McNeil Laboratories (Fort Washington, Pa.), Smith Kline Laboratories (Philadelphia, Pa.), Beiersdorf, A.G. (Hamburg, G e r m a n y ) a n d Ciba-Geigy (Summit, New Jersey). D-Amphetamine sulfate and apomorphine hydrochloride were obtained from the Medicinal Chemistry Section of Merck and Co. (West Point, Pa.).
3. Results
3.1. Antagonism of clonidine-induced enhancement o f the flexor reflex in the spinalized rat At the time of the first test, i.e. 30 min after treatment with clonidine, BE-2254 produced its greatest inhibitory effect (EDs 0 = 3.4 mg/kg i.p.) (fig. 1). The inhibitory action of BE-2254 subsided only gradually over time with the EDs 0 values at 60, 120 and 180 min being respectively, 3.6, 3.7 and 3.8 mg/kg. In contrast, the antagonistic action of chlorpromazine developed slowly with the ED50 values 30, 60, 120 and 180 min after treatment with clonidine being, respectively, 7.5, 5.1, 4.4 and 4.4 mg/kg i.p. (fig. 1). Haloperidol was essentially inactive at all time inter° vals and at all doses examined.
8 ~5
0
A I
3
9
mg/kg i.p.
Fig. 1. Antagonism of clonidine-induced enhancement of the flexor reflex in the spinalized rat. Pretreatment with vehicle, BE-2254, chlorpromazine (CPZ) or haloperidol was performed 30 min prior to treatment with clonidine, 1.2 mg/kg i.p. At each of the 3 pretreatment dose levels, the compounds were tested concurrently under random and blind conditions. The strength of the flexor reflex was determined 30, 60, 120 and 180 rain after administering clonidine. Values plotted in the figure are at time of greatest antagonism of clonidine, as follows: BE-2254 -- 30 rain, c h i o r p r o m a z i n e - 120 min, and haloperidol -- 60 rain after injecting clonidine. EDs0 values estimated from these data were: BE-2254, 3.4 (95% confidence interval = 2.9--3.9), chlorpromazine = 4.4 (4.3--4.6) and haloperidol >9 mg/kg i.p.
Phentolamine was tested at one dose level, 9 mg/kg i.p. 30 min prior to treatment, for inhibition of clonidine-induced enhancement of the flexor reflex. 10 rats received phentolamine + clonidine and a second 10 got vehicle + clonidine. Total scores (maximum total possible = 3 × n = 30) for phentolamine + clonidine at 30, 60, 120 and 180 min after injecting clonidine were 25, 26, 24 and 22, while in the vehicle-pretreated animals the total scores equaled 25, 27, 26 and 25. Phentolamine was, obviously, ineffective.
282
B.V. CLINESCHMIDT ET AL
z
°iO0
50 c.) c~
_z
BE-2
HALOPERI D
8 o
I
I
I
I
I
I
0.05
0.I
0.5
I
5
I0
50
Ioo
mg/kg i.p.
Fig. 2. Inhibition of apomorphine-induced gnawing in rats. 1 hr following pretreatment, the rats received apomorphine (3 mg/kg s.c.). 1 hr later, the animals were observed for the presence and degree of stereotyped gnawing. Details are given under Materials and methods. EDs 0 values estimated from these data were: haloperidol = 0.11 (95% confidence interval 0.11--0.11), chlorpromazine (CPZ) = 1.42 (1.24--1.37) and BE-2254 = 55.0 (48.2--62.2). TABLE 1 Elicitation of posturai asymmetries in mice with a unilateral striatal lesion. Treatment (mg/kg i.p.)
Methocel BE-2254
Methocel Haioperidol
Methocel Chlorpromazine
No.* contralateral asymmetries/No, tested 1 hr
2 hr
3 hr
(8.9) (13.3) (20.0) (30.0)
1/10 2/10 3/10 6/10 9/10 E D s 0 : 1 6 . 4 (12.4--21.6)**
1/10 0/10 1/10 3/10 4/10 > 30
0/10 0/10 0/10 0/10 0/10 > 30
(0.29) (0.47) (0.75) (1.2)
0/10 1/10 0/10 5/10 5/10 EDs 0 : --
0/10 0/10 2/10 7/10 9/10 0.64
0/10 2/10 3/10 8/10 9/10 0.54 (0.33--0.88)**
(1.5) (2.25) (3.38) (5.06)
0/10 1/10 1/10 5/10 10/10 EDs0:3.4
0/10 0/10 1/10 5/10 10/10 3.1 (2.1--4.6)**
0110 0/10 0/10 5/10 10110 3.4
* Deviations or circling to side opposite the lesion. Observations for asymmetries were carried out under random and blind conditions at 1, 2 and 3 hr after treatment. ** 95% confidence interval, enclosed in parentheses, is given for time of maximum effect.
BE-2254 A N D C A T E C H O L A M I N E R E C E P T O R B L O C K A D E
3.2. Antagonism gnawing in rats
of
283
ble 1). The action of BE-2254 was rather transient, whereas that of haloperidol developed gradually with time. Chlorpromazine appeared to be a b o u t equally effective at 1, 2 or 3 hr post injection.
apomorphine-induced
BE-2254 was considerably less p o t e n t (EDs0 = 55.0 mg/kg i.p.; 95% confidence interval = 48.2--62.2) than either chlorpromazine (EDs 0 = 1.42; 1.24--1.37) or haloperidol (EDs 0 = 0.11; 0.11--0.11) as an antagonist of apomorphine (fig. 2).
3. 4. Inhibition o f amphetamine-stimulated locomotor activity Stimulation of l o c o m o t o r activity in mice by amphetamine was virtually completely blocked by 5 mg/kg of BE-2254 or 0.5 mg/kg of haloperidol (table 2). BE-2254 (EDs0 = 1.6 mg/kg) was about 1/10th as potent as haloperidol (EDs0 = 0.18 mg/kg) as an antagonist of amphetamine.
3.3. Elicitation o f postural asymmetries in lesioned mice All three c o m p o u n d s elicited deviations or circling contralateral to the lesioned side (taTABLE 2
A n t a g o n i s m o f a m p h e t a m i n e - s t i m u l a t e d l o c o m o t o r activity in mice.
Pretreatmentt (mg/kg i.p.)
Treatmenttt (mg/kg s.c.)
Vehicle Vehicle BE-2254
(5) (5) (2.5) (2.5) (1.25) (1.25) (0.625) (0.625)
Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine
(0.5) (0.5) (0.25) (0.25) (0.125) (0.125) (0.0625) (0.0625)
Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine Vehicle Amphetamine
Vehicle Vehicle Haloperidol
Counts/1 hr (~-± S.D. of square roots)
(3) (3) (3) (3) (3)
(3) (3) (3) (3) (3)
42.2 84.9 *27.3 **29.5 *34.9 **45.7 38.0 **65.6 36.6 *'74.1
± ± ± ± ± ± ± ± ± ±
9.8 15.8 11.4 12.1 12.4 20.9 11.3 17.2 14.7 20.0
37.5 66.0 "16.0 *'21.0 "18.6 **29.8 *26.0 **43.2 *26.5 59.6
± ± ± ± ± ± ± ± ± ±
6.3 10.5 7.7 9.8 5.8 9.0 8.2 12.2 8.2 14.4
A m p h e t a m i n e ++ % Inhibition stimulated activity
42.7 2.2
94.6
10.8
74.6
27.6
35.3
37.5
12.2 EDs 0 = 1.61 (1.19--2.17) +
28.5 5.0
82.5
11.2
60.7
17.2
39.6
33.1 EDs 0 = 0.18 (0.16--0.19) +
t tt + ++ * **
n = 2 0 / g r o u p ; 30 (BE-2254) or 90 (Haloperidol) rain before t r e a t m e n t . 15 rain before i n t r o d u c t i o n into " d o n u t " - s h a p e d p h o t o c e l l units. 95% c o n f i d e n c e interval. ( P r e t r e a t m e n t + a m p h e t a m i n e ) -- ( p r e t r e a t m e n t + vehicle). C o m p a r e d with vehicle + vehicle C o m p a r e d w i t h vehicle + a m p h e t a m i n e ~ P < 0.05 (2-tailed t-test).
284
4. Discussion
In addition to stimulating s-receptors for norepinephrine in peripheral tissues (Hoefke and Kobinger, 1966), clonidine also stimulates receptors in the CNS having characteristics similar to the peripheral s-receptor (And~n et al., 1970; Kobinger and Walland, 1971; Schmitt et al., 1971). And~n (1970) has summarized the rather extensive evidence indicating that activation of noradrenergic receptors in the spinal cord leads to an enhancement of the flexor reflex. Antagonism of the increase in the flexor reflex elicited by clonidine thus provides a means for identifying centrally acting antagonists of receptors for norepinephrine. BE-2254 and chlorpromazine were effective in this regard, whereas phentolamine and haloperidol were not. Clonidine's action on the flexor reflex can also be inhibited somewhat with the s-blockers phenoxybenzamine and yohimbine, b u t high (~>10 mg/kg) doses are required (unpublished observations). Considering the differences between the experimental procedures, it seems rather interesting that our findings agree closely with results obtained from microiontophoretic studies on neurons in the brain stem of the cat. Chlorpromazine (Bradley et al., 1966) specifically antagonizes the excitatory action of norepinephrine on brain stem neurons, while phentolamine and phenoxybenzamine are essentially ineffective (Boakes et al., 1971). Apomorphine elicits stereotyped behavior, including compulsive gnawing, by a direct agonistic effect on central receptors for dopamine (Ernst, 1967; And~n et al., 1967). Inhibition of apomorphine-induced gnawing by BE-2254 suggested, therefore, an ability to act as a central dopamine receptor antagonist. Like the widely accepted antagonists of dopamine, chlorpromazine and haloperidol, BE2254 also produced postural asymmetries in mice with a unilateral lesion in the striatum. These findings were somewhat unexpected, since such an action of BE-2254 could hardly
B.V. C L I N E S C H M I D T ET AL.
have been predicted from the compound's chemical structure. An interesting difference was noted in the relative potency of BE-2254 in the two tests for dopamine receptor antagonism. BE-2254 was about 500 times less potent than haloperidol in the apomorphine antagonism test, but only approximately 30 times less active in the postural asymmetry test. Several explanations could be advanced, although two seem most likely. First of all, it could simply reflect a species difference, since the rat was used in one procedure and the mouse in the other. The time course of the blocking effect of BE-2254 on receptors for dopamine might also provide at least a partial explanation. As shown in the lesioned mouse, BE-2254's action was transient, falling off greatly between 1 and 2 hr post injection. Antagonism of apomorphine-evoked gnawing was determined only at one time, 2 hr after pretreatment with BE-2254 or one of the other compounds. Therefore, assuming transient dopamine receptor blockade in the rat as well, the effect of BE-2254 may have greatly subsided by 2 hr following administration, while the longer acting haloperidol was still quite effective. In any event, our observations clearly demonstrate the central dopamine receptor blocking action of BE-2254, and it is not clear why Yehuda and Wurtman (1974) did not detect this effect of BE-2254 in the rat, using either the apomorphine stereotypy test or the rotational behavior model. Although BE-2254 resembles chlorpromazine, inasmuch as it appears to block central receptors for either norepinephrine or dopamine, BE-2254 also differs importantly from chlorpromazine. BE-2254 has little, if any, peripheral (and presumably central) antiserotonin activity (Gothert et al., 1973; Baumgarten et al., 1972}. Moreover, unlike chlorpromazine, BE-2254 does not cause any significant dilation of the mouse's pupil in doses up to 30 mg/kg i.p. (unpublished observations), suggesting a lack of any substantial anticholinergic activity. As the data reported herein would indicate, BE-2254 is a b o u t comparable
BE-2254 AND CATECHOLAMINE RECEPTOR BLOCKADE
to chlorpromazine as an antagonist of central receptors for norepinephrine, but it is a weaker agent with respect to receptors for dopamine. Considering the above, BE-2254 may prove to be a useful pharmacological agent for producing blockade of central receptors for norepinephrine. It is generally agreed that amphetamine stimulates locomotor activity in mice and rats by releasing catecholamines from monoaminecontaining neurons in the CNS. However, there is no agreement concerning the relative importance of dopamine and norepinephrine, and findings reported by several authors suggest that both amines are involved (Corrodi et al., 1970; Svensson, 1970; Rolinski and ScheelKriiger, 1973). Inhibition by BE-2254 of amphetamine-stimulated locomotor activity in the mouse provides additional indirect evidence that this compound antagonizes central catecholamine receptor-dependent responses. Based on the evidence provided in this study, it seems likely that BE-2254 antagonizes amphetamine by virtue of its receptor blocking action, although there is no evidence for excluding other possible mechanisms, e.g. inhibition of catecholamine release. To summarize, indirect evidence has been obtained indicating that BE-2254 administered systemically blocks central receptor sites for catecholamines and impairs catecholamine receptor-mediated responses. The norepinephrine receptor blocking action of BE2254 is roughly comparable to that of chlorpromazine, whereas BE-2254 is considerably less active than chlorpromazine or haloperidol as an antagonist of receptors for dopamine. Although additional evidence is admittedly needed in order to establish the selectivity of BE-2254, it appears that this agent is useful for producing blockade of central receptors for norepinephrine.
References And~n, N.-E., 1970, Effects of amphetamine and some other drugs on central catecholamine mecha-
285
nisms, in: International Symposium on Amphetamines and Related Compounds, eds. E. Costa and S. Garattini (Raven Press, New York) p. 447. And~n, N.-E., H. Corrodi, K. Fuxe, B. HSkfelt, T. H6kfelt, C. Rydin and T. Svensson, 1970, Evidence for a central noradrenaline receptor stimulation by clonidine, Life Sci. 9, Part I, 513. And~n, N.-E., A. Rubenson, K. Fuxe and T. HSkfelt, 1967, Evidence for dopamine receptor stimulation by apomorphine, J. Pharm. Pharmacol. 19, 627. Baumgarten, H.G., M. Gothert, H.G. Schlossberger and P. Tuchinda, 1972, Mechanism of pressor effect of 5,6-dihydroxytryptamine in pithed rats, Naunyn-Schmiedeb. Arch. Pharmakol. 274, 375. Benthe, H.F., M. Gothert and P. Tuchinda, 1972, Noradrenalin-antagonistische Wirkung verschiedener Phenylaethylamin- und PhenoxyaethylaminDerivate, Arzneim. Forsch. 22, 1468. Boakes, R.J., P.B. Bradley, N. Brookes, J.M. Candy and J.H. Wolstencroft, 1971, Actions of noradrenaline, other sympathomimetic amines and antagonists on neurones in the brain stem of the cat, Brit. J. Pharmacol. 41,462. Bradley, P.B., J.H. Wolstencroft, L. Hosli and G.L. Avanzino, 1966, Neuronal basis for the central action of chlorpromazine, Nature, London 212, 1425. Corrodi, H., K. Fuxe, A. Ljungdahl and S.-O. Ogren, 1970, Studies on the action of some psychoactive drugs on central noradrenaline neurones after inhibition of dopamine-~-hydroxylase, Brain Res. 24, 451. Ernst, A.M., 1967, Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats, Psychopharmacol. 10, 316. Gothert, M., P. Tuchinda and H.G. Baumgarten, 1973, Pressor effect of indolethylamines, European J. Pharmacol. 21,242. Hoefke, W. and W. Kobinger, 1966, Pharmakologische Wirkung des 2--(2,6--Dichlorophenylamino)2-Imidazolin-hydrochjorids, einer neuen, antihypertensiven Substanz, Arzneim. Forsch. 16, 1038. Kobinger, W. and A. Walland, 1971, Involvement of adrenergic receptors in central vagus activity, European J. Pharmacol. 16, 120. Lotti, V.J., 1971, Action of various centrally acting agents in mice with unilateral caudate brain lesions, Life Sci. 10, 781. Rolinski, Z. and J. Scheel-Kriiger, 1973, The effect of dopamine and noradrenaline antagonists on amphetamine-induced locomotor activity in mice and rats, Acta Pharmacol. Toxicol. 33, 385. Schmitt, H., H. Schmitt and S. F4nard, 1971, Evidence for an a-sympathometic component in the e f f e c t s of catapressan on vasomotor centres: antagonism by piperoxane, European J. Pharmacol. 14, 98.
286 Svensson, T.H., 1970, The effect of inhibition of catecholamine synthesis on dexamphetamine-induced central stimulation, European J. Pharmacol. 12, 161. Yehuda, S. and R.J. Wurtman, 1974, Paradoxical
B.V. CLINESCHMIDT ET AL thermoregulatory behavior in rats induced by (+) amphetamine: blockade by ~-adrenoceptor or dop amine receptor blocking agents, J. Pharm. Pharma col. 26, 210.